The present invention provides phosphonate-substituted dyes, including rhodamines, rhodols and fluoresceins that are additionally substituted with 3-amido group. The dyes of the invention, including chemically reactive dyes and dye-conjugates are useful as fluorescent probes, particularly in biological samples.
Xanthenes are among the most commonly used dyes in biological applications where a highly sensitive detection reagent is required. All carboxyphenyl-substituted xanthene dyes belong to three basic structures: fluorescein, rhodamine and rhodol.

“Fluorescein” dyes include derivatives of 3H-xanthen-6′-ol-3′-one that are typically substituted at the 1-position by an ortho-carboxyphenyl group (hereinafter a 3-carboxyphenyl group, consistent with the numbering above). “Rhodamine” dyes include derivatives of 6′-amino-3H-xanthen-3′-imine that are typically substituted at the 1-position by a 3-carboxyphenyl group. “Rhodol” dyes include derivatives of 6′-amino-3H-xanthen-3′-one that are typically substituted at the 1-position by a 3-carboxyphenyl group. The 3-carboxy group can exist in a free ionized, free protonated or a spiro-lactone form depending on solvent and acidity.
Xanthene dyes have a polycyclic aromatic nature and are generally hydrophobic. Those molecules are also prone to minimize exposure to any hydrophilic environment through interactions with nearby hydrophobic surfaces and residues. These interactions include dye-dye interaction and dye-biomolecule (e.g. proteins, lipids, oligonucleoties) interactions. Hydrophobic interactions can cause substantial quenching effects for fluorescent dyes (see for example Randolph, J. B.; Waggoner, A. S. Nucleic Acids Res. 1997, 25(14), 2923-2929 and references cited therein). One method to overcome this problem is to improve the hydrophilic character of the dye by introducing a phosphonate substituent into the dye molecule as disclosed in U.S. application 2006/0199955. Alternatively, sulfonate-substituted dyes can also be utilized as disclosed in U.S. Pat. Nos. 5,268,486 and 6,130,101.
The phosphonate groups disclosed in U.S. application 2006/0199955 have certain advantages over the sulfonate groups. For instance, they do not merely provide the desired negative charge but also introduce functional groups suitable for conjugation with biological agents. However, the activation of the functional groups such as COOH, OH (which requires treatment with acid chlorides), anhydrides, activated esters or various dehydrating agents, is complicated by the concurrent activation of the 3-carboxy group (see U.S. Pat. No. 6,750,357, and U.S. patent application 2006/0154251). The undesired side reaction precludes or significantly reduces the formation of the target mono-activated dyes. Another example of a side reaction involving the 3-carboxy group is described in Lyttle et al. J. Org. Chem. 2000; 65(26): 9033-9038. In this example the spiro-lactone form of a rhodamine dye is shown to react with tert-butylamine used for the oligonucleotide deprotection thus generating a 3-carboxamide side product.
It is therefore an object of the present invention to modify phosphonate-substituted xanthene dyes in such a way that the 3-carboxy group may not be capable of undesired side reactions. This object is achieved by substituting the 3-carboxy group with 3-amido group.
The present invention therefore provides phosphonate-substituted dyes, including rhodamines, rhodols and fluoresceins that are additionally substituted with 3-amido group. The xanthene dyes of the invention possess significant advantages over their carboxy-substituted analogs as well as the non-phosphonylated 3-amidophenyl-xanthenes disclosed in U.S. Pat. Nos. 4,290,955; 4,647,675; 6,399,392; 6,750,357, and U.S. Patent Application No. 2006/0154251, as well as PCT publications WO 2002/055512 and WO 2005/102176. The phosphonate group provides both conjugation capability and additional hydrophilicity to the dye molecules, while the 3-amido substituent blocks undesired side reactions. Overall, the compounds provided herein exhibit increases in aqueous solubility and further exhibit reduced aggregation.